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THP-1 cells treated with plasticizer DINCH and metabolite MINCH

ABSTRACT: Since 2008, the consumption of the plasticizer bis (2-ethylhexyl) phthalate (DEHP) in the manufacture of plastic products has been restricted due to several adverse health effects. 1,2-cyclohexanedicarboxylic acid diisononyl ester (DINCH) is increasingly used as a harmless substituent for DEHP. However, the structural similarity to DEHP and first studies demonstrating adverse effects on reproduction and metabolism in rats, suggest that DINCH may act adverse on human health. Still, there is a lack of studies on the biological effects of DINCH, especially in the immune system, which is in constant communication with other tissues and thus crucial for maintaining a functional organism. Here we aimed to investigate the effects of DINCH and its naturally occurring biotransformation product monoisononylcyclohexane-1,2-dicarboxylic acid ester (MINCH) on the innate immune response. Human THP-1 macrophages were exposed to DINCH and MINCH in a concentration range of 10 nM to 10 μM for 4 h, 16 h and 24 h in the presence of LPS.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Suspension Culture, Macrophage

DISEASE(S): Acute Leukemia

SUBMITTER: Alexandra Schaffert

LAB HEAD: Kristin Schubert

PROVIDER: PXD027744 | Pride | 2022-01-10

REPOSITORIES: Pride

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Source:

			Action	DRS
	20190306_Uniprot_HomoSapiens_Refset_IK.fasta	Fasta
	20200730_THP1_DINCH.mzML	Mzml
	20200730_THP1_DINCH.mzid.gz	Mzid
	THP1_DINCH_10_1.raw	Raw
	THP1_DINCH_10_2.raw	Raw

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Project description:Bioactivities of fungal peptides are of interest for basic research and therapeutic drug development. Some of these peptides are derived from “KEX2-processed repeat proteins” (KEPs), a recently defined class of precursor proteins that contain multiple peptide cores flanked by KEX2 protease cleavage sites. Genome mining has revealed that KEPs are widespread in the fungal kingdom. Their functions are largely unknown. Here, we present the first in-depth structural and functional analysis of KEPs in a basidiomycete. We bioinformatically identified KEP-encoding genes in the genome of the model agaricomycete Coprinopsis cinerea and established a detection protocol for the derived peptides by overexpressing the C. cinerea KEPs in the yeast Pichia pastoris. Using this protocol, which includes peptide extraction and mass spectrometry with data analysis using the search engine Mascot, we confirmed the presence of several KEP-derived peptides in C. cinerea as well as in the edible mushrooms Lentinula edodes, Pleurotus ostreatus and Pleurotus eryngii. Processing by the dipeptidyl aminopeptidase STE13 is likely involved in the biosynthesis of some of these peptides. While CRISPR-mediated knockouts of the C. cinerea kep genes did not result in any detectable phenotype, knockout of kex genes caused defects in mycelial growth and fruiting body formation. These results suggest that KEP-derived peptides may play a role in the interaction of C. cinerea with the biotic environment and that the KEP-processing KEX proteases target a variety of substrates in agaricomycetes, including some important for mycelial growth and differentiation. In the past, fungal RiPPs and KEP-derived peptides were mostly studied using forward genetics, where the peptides were first isolated and characterized and only then the corresponding precursor genes were identified (e.g., ustiloxins, phomopsins, candidalysin, Rep1, omphalotin). In this paper, we established a protocol for the identification of KEP-derived peptides in fungal samples by a combination of reverse genetics and peptidomics. The protocol was developed using the model agaricomycete Coprinopsis cinerea, but is also applicable to other fungi. In a first step, we bioinformatically screened the predicted proteome of C. cinerea for KEP-encoding genes. Second, we expressed six different KEP genes from C. cinerea in the yeast Pichia pastoris to (1) establish a protocol for extraction and detection of KEP-derived peptides by mass spectrometry in P. pastoris culture supernatants known for their low complexity in terms of proteins and peptides (Higgins 1995); (2) obtain an indication that the detected KEPs were indeed processed to peptides, and (3) use the structures of the detected peptides as a guide for which peptides to expect in C. cinerea in terms of peptide length and peptide modifications. In a third step, we applied the established peptide extraction and detection method to detect KEP-derived peptides in culture supernatants and tissue samples of the agaricomycetes C. cinerea, Lentinula edodes, Pleurotus ostreatus and Pleurotus eryngii.

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THP-1 cells treated with plasticizer DINCH and metabolite MINCH

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